Eclipse te200 confocal microscope

At 128 days of mouse age, the mice were transcardially perfused with normal saline followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for fixation. Spinal cord portions (L2-L4) were removed, post-fixed in 4% PFA overnight at 4℃, and transferred to 30% sucrose. The tissues were then embedded into a mold filled with optimum cutting temperature (OCT) compound (Tissue-Tek O.C.T Compound; Sakura Finetek, Japan) and stored at −80℃ before cryosectioning. The spinal cords were serially cut on a cryostat into 10 µm thick cross-sections and washed in PBS to remove the OCT compound. After the sections were blocked with 3% to 5% normal goat serum in PBS at room temperature, they were incubated with the primary antibody overnight at 4℃. For detection of the primary antibody, sections were incubated with the appropriate Alexa 488- or 594-conjugated secondary antibodies (1:2,000; Molecular Probes, USA) for 1 h in the dark at room temperature. Images were captured with an Eclipse TE200 confocal microscope (Nikon). To determine the relative immunohistochemical intensity of each signal, ten spinal cord sections per mouse were used (n = 4–5 per group). Each signal was measured in equivalent areas in the anterior horn of the lumbar spinal cord by using ImageJ v. 1.63 software (National Institutes of Health).

For hematoxylin and eosin (H&E) staining, cryotissues were sectioned at 7 to 8 µm thickness and then stained with H&E by using a standard protocol [9 (link)]. The detection of Nissl bodies in the cytoplasm of neurons was performed by Nissl staining. Briefly, the tissues were incubated in 1:1 alcohol/chloroform solution overnight and then immersed in 100% alcohol followed by 95% alcohol and dH₂O for rehydration. The rehydrated tissues were stained with 0.1% cresyl violet (Sigma-Aldrich, Germany), rinsed, and then slide mounted with coverslips. Fluoro-Jade C (FJC) staining was used to identify degenerating neurons in the CNS. First, the tissues were immersed in 100% alcohol and then 70% alcohol for 3 min each. The tissues were then transferred to 0.06% KMnO₄ for 15 min, washed with dH₂O, and stained with a 0.001% FJC solution for 30 min in a dark room. After staining, the slides were rinsed again and mounted with coverslips. Counting Nissl stained or FJC stained motor neurons was performed in equivalent size frames in an anterior horn region, and ten samples were counted per mouse (n = 4–5 per group). All histological examinations were performed by using an Eclipse TE200 confocal microscope (Nikon).

Assembloids or organoids were fixed in 4% paraformaldehyde overnight at 4℃, dehydrated in 30% sucrose solution and embedded in OCT compound (Tissue Tek, Germany). Cryosections were cut into 10 μm thick sections, washed with phosphate-buffered saline (PBS), deparaffinized, rehydrated and stained with hematoxylin/eosin (HE) and picrosirius red.
For immunohistochemistry, cryosections were cut into 20 μm thick sections and mounted onto silane-coated slides (Muto, Japan). Samples were washed 3 times with PBS, blocked and permeabilized for 1 h at room temperature (RT) in 5% normal goat serum and 0.5% Triton X-100 diluted in PBS. Primary antibodies were incubated overnight at 4℃ followed by incubation with appropriate secondary antibodies and 4’,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Samples were mounted using mounting media and then dried at RT overnight. All samples were imaged using an Eclipse TE200 confocal microscope (Nikon, Japan).